Hepatitis C virus infects 4 million Americans and an estimated 2-4% of the world's population, causing chronic hepatitis in most of those infected. A sizable fraction subsequently develops cirrhosis or hepatocellular carcinoma. This proposal describes the use of a novel approach to perform a mutational analysis of regions of the Hepatitis C virus (HCV). The objective is to delineate, at an unprecedented resolution, the role of specific sequences, both viral and cellular, in viral entry. Conventional approaches to the study of viral entry consist of morphological and biochemical studies of replication intermediates. While these approaches have been very useful for many viruses, they suffer from some drawbacks. Since viral entry is an inefficient process, with only one out of hundreds or thousands of virions following the productive pathway of infection, the non-productive particles tend to severely obscure morphological analysis and limit interpretation of biochemical studies. It also becomes difficult to get an adequate signal with physiologically relevant multiplicities of infection. This is especially pertinent to the study of the early steps of viral infection, before there is any amplification from viral expression. These problems make it necessary to complement conventional studies with the genetic approach of making and analyzing mutations that disrupt viral functions. The mutational approach is a proven and well-established strategy for the study of gene function. However, most current methods involve the isolation, storage and characterization of each mutant separately, making the process very time consuming and labor intensive. Genetic footprinting is a novel method that allows for efficient construction and parallel functional analysis of thousands of mutations in a cloned gene. The specific aims of this research are: 1) Creation of libraries of mutations in the E1 and E2 envelope glycoproteins of HCV, using a transposon-based mutagenesis method, followed by their analysis. 2) Genetic footprinting of the HCV IRES, to determine regions necessary for its function. Preliminary data showing the successful generation of libraries is presented. These libraries will be analyzed en masse to determine what regions of the glycoproteins are necessary for viral binding and fusion, and to determine what regions of the IRES are essential for translation of viral proteins. A comprehensive and detailed knowledge of the process of HCV entry gained from this study will be important for understanding the mechanism of viral infection and for the development of new preventive and therapeutic approaches against HCV.